Diagnosis of meningitis in immuno-compromised hosts, such as those with AIDS or cancer, is difficult because of the lack of sensitive methods for detecting the most likely pathogens. Organisms in these settings, such as cytomegalovirus (CMV), herpes simplex virus (HSV), and varicella-zoster virus (VZV), are difficult to grow from cerebrospinal fluid, and no good antigen detection techniques are available. Over the past few years, it has become increasingly clear that detecting these viral agents by the polymerase chain reaction (PCR) method may provide the most sensitive and specific diagnostic tests. Therefore, we are developing a battery of in-house PCR assays for CMV, HSV, and VZV so that they will be available as routine service tests for CSF from NIH patients. The PCR assay for CMV in spinal fluid has been completely developed, and validated and is being offered as a routine molecular diagnostic assay. The HSV and VZV assays are being developed with the same performance characteristics as the existing CMV protocol so that simultaneous detection of all three viruses can be accomplished in a single assay run. All three assays utilize internal mimics to evaluate the efficiency of amplification within individual PCR tubes and to detect PCR inhibition, which will eliminate this source of false negative results. Endpoint detection by europium-labeled fluorimetric hybridization probes allows a lower limit of detection of 3 to 5 viral genome equivalents. Evaluations of the CMV and HSV assays show excellent specificity.